Yersinia pestis, the causative agent of pneumonic and bubonic plague, is considered a potential bioterrorism agent. The bacterium is easy to grow and genetically manipulate and can be delivered in aerosolized droplets. The resulting pneumonic plague has a short incubation time and is rapidly and highly fatal. Great potential exists for spread of pneumonic plague from primary infected individuals to their contacts. Currently, there is no available vaccine against pneumonic plague. While much is known concerning the virulence determinants necessary for Y. pestis to cause disease by subcutaneous and intravenous routes of infection, essentially nothing is known about the virulence factors required for pneumonic plague. Such factors would be potential candidates for a subunit vaccine or targets for therapeutic agents designed to protect against pneumonic plague. Therefore the overall goal of this research application is to identify determinants important for the virulence of Y. pestis in a mouse model of pneumonic plague. The pH6 antigen is a putative adhesin, which is required for full virulence of Y. pestis by an intravenous route of infection, while different iron transport systems are important in the pathogenesis of bubonic and septicemic plague in mice. Recently we have discovered that a heme transport system, Hmu, is necessary for the optimal growth of Y. pestis in a macrophage cell line. In Specific Aim 1, we will use Y. pestis strains carrying specific mutations in the pH6 antigen and select iron/heme transport systems to test the potential role of these systems in the virulence of pneumonic plague. In Specific Aim 2, signature-tagged mutagenesis will be used to identify additional gene products required for the pathogenesis of pneumonic plague. These studies will enhance our understanding of the factors important in the pathogenesis of pneumonic plague and facilitate the development of measures to protect people against possible bioterrorism based on Y. pestis.